P Placentalexpressionofaminopeptidase-Q(laeverin)anditsroleinthepathophysiologyofpreeclampsia Research

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OBSTETRICS

Placental expression of aminopeptidase-Q (laeverin) and its role in the pathophysiology of preeclampsia

Mona Nystad, MSc; Vasilis Sitras, MD, PhD; Merethe Larsen, MSc; Ganesh Acharya, MD, PhD

OBJECTIVE: The purpose of this study was to investigate the expression and subcellular localization of laeverin, a placenta-specific membrane-bound aminopeptidase, in preeclamptic placentas and its role in trophoblast cell migration and invasion.

STUDY DESIGN:Expression of laeverin was investigated in 6 normal and 6 preeclamptic placentas with the use of immunofluorescence, sodium dodecylsulfate-polyacrylamide gel electrophoresis with Western blot analysis and immunoelectron microscopy. The role of laeverin in trophoblast migration and invasion was studied with the use of the xCelligence system and Boyden chambers with Matrigel in HTR-8/SVneo cells. The effect of laeverin gene-silencing on selected genes that are involved in cell transformation and tumorigenesis was evaluated by polymerase chain reaction array. The Studentttest, Mann-WhitneyU test,

c

²test, or F-test was used to compare groups as appropriate.

RESULTS:Laeverin was expressed in the cell membrane of villous trophoblasts in third-trimester healthy placentas; in preeclamptic placentas, it was expressed ectopically in the cytoplasm, especially in

microvesicles. Immunoelectron microscopy showed laeverin leakage into the fetal capillaries and abundant expression in microvesicles in preeclamptic placentas. Migration and invasion of HTR-8/SVneo cells were reduced by 11.5% (P¼.023) and 56.7% (P¼.001), respectively, by laeverin geneesilencing. Analysis of downstream pathways affected by laeverin-silencing demonstrated significant down- regulation of integrin A2 (39-fold), integrin B3 (5-fold), and matrix metalloprotease 1 (36-fold).

CONCLUSION:Expression of laeverin protein is altered in preeclamptic placentas. Its ectopic expression in the cytoplasm and microvesicles, rather than the cell membrane and leakage into the fetal capillaries, may have a role in the pathophysiologic condition of preeclampsia.

Laeverin gene appears to be involved in trophoblast cell migration and invasion through interaction with integrins and matrix metalloprotease 1.

Key words:aminopeptidase, laeverin, microvesicle, placenta, preeclampsia

Cite this article as: Nystad M, Sitras V, Larsen M, et al. Placental expression of aminopeptidase-Q (laeverin) and its role in the pathophysiology of preeclampsia. Am J Obstet Gynecol 2014;211:686.e1-31.

P

reeclampsia complicates 5-10% of pregnancies and is a major cause of maternal mortality worldwide.¹Although it is clearly a placenta-speciﬁc disorder, its pathogenesis is not understood fully.

Therefore, its prediction, timely diagnosis, and appropriate management remain challenging.

Laeverin, a membrane-bound aminopeptidase, was ﬁrst reported to be expressed by human trophoblast cells

in 2004 by Fujiwara et al²and has been suggested to cooperate with the chemokine system in the regulation of human placentation.³ The same group recently presented some molecular evi- dence suggesting that laeverin is important for extravillous trophoblast invasion.⁴ Laeverin is a trophoblast- speciﬁc protein; however, it has been reported to be expressed in other tissues in some inﬂammatory diseases, such as

rheumatoid arthritis.⁵ In a previous study, comparing global placental gene expression proﬁle between preeclamptic and healthy pregnancies, we found 16 genes that were able to predict preeclampsia phenotype in our study population.⁶ Laeverin was among those genes, and it was up-regulated signiﬁ- cantly in the preeclamptic placentas.

Therefore, we hypothesized that the deregulation of laeverin protein may lead

From the Department of Clinical Medicine, Women’s Health and Perinatology Research Group, Faculty of Health Sciences, University of Tromsø, Norway (Ms Nystad and Drs Sitras and Acharya), and Departments of Obstetrics and Gynecology (Ms Nystad and Drs Sitras and Acharya) and Occupational and Environmental Medicine (Ms Larsen), University Hospital of North Norway, Tromsø, Norway; Department of Obstetrics, Oslo University HospitaleRikshospitalet, Oslo, Norway (Dr Sitras); and Department of Clinical Sciences, Intervention, and Technology, Karolinska Institute, Stockholm, Sweden (Dr Acharya).

Received March 14, 2014; revised May 26, 2014; accepted June 18, 2014.

Supported by Northern Norway Regional Health Authority grant numbers 12032 and 12101 and by the Division of Child and Adolescent Health, University Hospital of North Norway, through its 2012 Research Fund.

The authors report no conﬂict of interest.

Corresponding author: Mona Nystad, MSc.mona.nystad@unn.no

686.e1 American Journal of Obstetrics&Gynecology DECEMBER 2014

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to abnormal trophoblast function and have a role in the pathophysiologic condition of preeclampsia.

In the present study, we investigated the expression of laeverin protein and its subcellular localization in healthy and preeclamptic placentas. Additionally, we investigated the role of laeverin in trophoblast cell migration and invasion.

M

ATERIALS AND

M

ETHODS

The study was approved by the Regional Committee for Medical and Health Research Ethics-North Norway (REK Nord reference no. 2010/2058-4), and informed written consent was obtained from all the participants.

Placental samples from a total of 12 pregnant white European women (6 healthy and 6 with severe preeclampsia) were collected. They were matched for maternal age, parity, onset of labor (spontaneous or induced), and the mode of delivery (vaginal or cesarean), and the results were compared.Severe preeclamp- siawas deﬁned as blood pressure160/

110 mm Hg and proteinuria 300 mg/

24-hour urine or2þin spot urine after 20 weeks of gestation in previously normotensive women.⁷ Women with preexisting medical conditions that may have affected the course and outcome of pregnancy were excluded. Doppler ultra- sonography was performed at48 hours before delivery in each case to assess ute- roplacental and umbilical circulation.

Collection of maternal blood and placental samples

Maternal venous blood samples were taken at 48 hours before delivery for the analysis of hemoglobin, hematocrit, liver function, and renal function. None of the women were in labor when blood samples were taken. Placental tissue samples were obtained immediately after delivery, as described previously.⁶

Cell line

The immortalized HTR-8/SVneo trophoblast cell line, which was obtained from primary cultures of human trophoblast cells,⁸ was used for migration, invasion, and polymerase chain reaction (PCR) array studies. Cells were maintained in RPMI Medium 1640

supplemented with 5% fetal bovine serum (FBS; GIBCO, Invitrogen, Carls- bad, CA) in a 37C water-jacketed incu- bator (Forma Scientiﬁc, Marietta, OH) with 5% CO2. Trypsin-ethylenediamine tetraacetic acid (Sigma Chemical Co, St.

Louis, MO) was used for harvesting and for the subculturing of cells.

Laeverin antibodies

Polyclonal antibodies against laeverin were raised (Eurogentec, Seraing, Belgium). Rabbits were immunized with synthetic oligo-peptides that contained 2 predicted epitopes (EP073418:CRV- HANLQTIKNENLK and EP073419:

CERAEVRGPLSPGTG). Peptide se- quences for these epitopes were chosen from the amino acid sequence of laeverin (Q0P5U8;http://www.ncbi.nlm.nih.gov/

protein/121946569). Immunogenic epitopes of the exposed amino acids of the laeverin 3-dimensional structure were chosen for peptide synthesis with software that was provided by Sigma Chemical Co.

A commercially available goat polyclonal antibody of laeverin was used as control (Santa Cruz Biotechnology Inc, Santa Cruz, CA).

Immunofluorescence

Tissue samples from 3 preeclamptic placentas and 3 normal healthy controls were ﬁxed in formalin, embedded in parafﬁn blocks, cut (4-6

m

sections), and mounted on glass slides. Immunoﬂuo- rescence cell staining was performed⁹ with our laeverin antibody (2.1

m

^g/mL)

and secondary goat anti-rabbit immunoglobulin G-ﬂuorescein isothiocyanate (2.5

m

g/mL; Santa Cruz Biotechnology Inc). Slides were counterstained with DAPI (4⁰,6-diamidino-2-phenylindole) II (Vysis; Abbott Diagnostics, Lake For- est, IL). Images were obtained with CytoVision digital system (Applied Im- aging, Grand Rapids, MI) that was equipped with a charge-coupled device camera (Cohu Inc, Poway, CA). A total of 200 cells were inspected on each slide. Experiments were run in triplicate.

Protein isolation

Placental tissue was cut in small pieces, and proteins were isolated with the

use of T-PER (Pierce Chemical Co, Rockford, IL) with Complete Mini ethylenediamine tetraacetic acidefree protease inhibitor cocktail in combina- tion MagNA Lyser Green Beads for homogenizing on MagNA Lyser (Roche, Indianapolis, IN). Protein concentration was measured with the use of the DC Protein Assay kit (Bio-Rad Labora- tories, Hercules, CA) in a ThermoMax Microplate Reader (Molecular Devices, Downington, PA).

Sodium dodecylsulfate-

polyacrylamide gel electrophoresis and Western blot analysis

Reduced and denatured proteins (5

m

^g)

that had been isolated from 8 placentas (4 preeclamptic and 4 normal) were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% NuPAGE (Invi- trogen). Electrophoresis and blotting (polyvinylidene diﬂuoride nylon membrane, pore size 0.45

m

m; Invitrogen) were run on Novex Mini Cell XCell Sure Lock (Invitrogen). Blots were cut under a 49-kDa protein band to provide 2 blots; 1 for laeverin and another for the housekeeping protein actin. Label- ing was done with primary antibodies against laeverin (our antibody [0.42

m

^g/

ml] and commercial antibody [1

m

^g/mL;

Santa Cruz Biotechnology Inc] or actin [1

m

g/mL; Santa Cruz Biotech- nology Inc]). Detection was performed with goat anti-mouse immunoglobulin G-alkaline phosphataseeconjugated antibody (0.2

m

^g/mL; ^Santa ^Cruz

Biotechnology Inc) and CDP-Star (Roche). Pictures were taken on Image- Quant LAS 4000 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Experiments were run in triplicate.

Immunoelectron microscopy

Immunoelectron microscopy was performed on ultrathin tissue sections of 2 healthy placentas and 2 placentas that were obtained from women with severe preeclampsia. All experiments were run in triplicates.

Fresh placental tissue samples were dissected, mounted in membrane car- riers, and frozen at high pressure (EMPACT 2 HPF; Leica Microsystems,

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DECEMBER 2014 American Journal of Obstetrics&Gynecology 686.e2

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Vienna, Austria). Frozen samples freeze substituted (EM AFS2, Leica Micro- systems) and infiltrated in Lowicryl HM20 (Electron Microscopy Sciences, Hatfield, UK).¹⁰Ultrathin sections of 70 nm were cut on a Leica EM UC6 ultra- microtome (Leica Microsystems) and mounted on copper grids (Agar Scien- tific, Stansted, UK) with Formwar and carbon.

Immunolabeling was performed with the optimal dilution of primary antibodies.^11,12 Single and double labeling experiments were performed with both locally designed (26.25

m

^g/

mL) and commercially purchased laeverin (5

m

g/mL) antibodies. For double labeling, anti-endoplasmatic reticulum (ER) mouse monoclonal antibody (RL90) to protein disulphide isomerase (ab2792; 0.1

m

g/mL) and anti-Golgi apparatus (GA) mouse monoclonal antibody (AE-6) to MG160 protein (MG160; ab58826; 0.05

m

g/mL; Abcam, Cambridge, UK) were used as the speciﬁc markers of ER and GA, respectively.

Microscopy was done with a JEM- 1010 transmission electron microscope (JEOL, Tokyo, Japan) at 4000, 10,000, 20,000, 30,000, and 70,000 magniﬁca- tions. Images were taken and processed in Morada Soft Imaging Camera system with iTEM software (Olympus, Hamburg, Germany). A total of 200 images from each experiment were processed. Image montage was done in Adobe Photoshop and Adobe Illustrator (Adobe Systems Inc, San Jose, CA).

Immunoglobulin G conjugated gold particles were used as controls in similar experiments. Possible secondary antibody cross-reactivity was excluded by the omission of primary antibodies in separate experiments.

xCelligence migration assay

HTR-8/SVneo trophoblast cells (210⁵ cells/well) were seeded the day before small interfering RNA (siRNA) transfection with FuGENE transfection reagent (Promega Corp, Madison, WI).

SiRNA (10 pmol) against laeverin or scrambled siRNA A or D (control; Santa Cruz Biotechnology Inc) were used.

Plates were incubated at 37C with 5%

CO2 for 5 hours; transfection solution

was replaced with fresh RPMI Medium 1640 with 5% FBS, and cells were further incubated for 24 hours; 210⁵cells/well were added to each well of the CIM-Plate 16 (ACEA Biosciences Inc, San Diego, CA). Migration assays were performed (for 72 hours, with sweeps of 30 minutes each) in the xCelligence system (ACEA Biosciences Inc). Three different CIM- Plates 16 were used. Experiments were run in quadruplicate. Coefﬁcients of variation for siRNA A, D, and laeverin were 3.5%, 1.2%, and 3.5%, respectively.

Untransfected cells were used as controls and were run in duplicates on each plate.

Analysis was performed in the RTCA software (version 1.2.1; ACEA Bio- sciences Inc).

Reverse transcriptionePCR of the cell line that was used in migration assays

Total RNA was isolated from cultured cells (untransfected, siRNA silenced laeverin siRNA A and D silenced) with TRIzol Reagent (Invitrogen) 55 hours after transfection at the migration opti- mum. Total RNA was extracted with RNeasy Mini Kit (Qiagen, Venio, The Netherlands); the concentration of RNA was measured with NanoDrop (Saveen Werner, Malmo, Sweden), and reverse transcription was performed with the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA). Complementary DNA samples were proﬁled for the relative expression of the genes of laeverin, glyceraldehyde- phosphate dehydrogenase, and actin, beta with the Taq Man Gene Expression Assays on 7900HT Fast Real-Time PCR system (Applied Biosystems).

Matrigel invasion studies in Boyden chambers

HTR-8/SVneo trophoblast cells (5 10⁵cells) were grown in RPMI Medium 1640 with 10% FBS and incubated at 37C, with 5% CO2overnight. Medium was replaced by RPMI Medium 1640 with 5% FBS the next day. On day 3, transfection with 50 pmol laeverin siRNA or 50 pmol of siRNA A control (Santa Cruz Biotechnology Inc) with Lipofectamine 2000 (Invitrogen) was performed in separate ﬂasks. Cultures

were incubated at 37C with 5% CO2

for 4 hours and washed with RPMI Medium 1640 without serum before in- cubation overnight. Invasion studies (1 10⁵ cells/well; 5% FBS used as chemoattractant) were performed in 48 hours in BD BioCoateBD Matrigel Invasion Chambers (24-well plate 8

m

with control inserts; BD Biosciences, San Jose, CA) and incubated at 37C with 5% CO2. The noninvading cells were removed from the upper part of the insert’s membrane by scrubbing with cotton-tipped swabs that had been moistened with medium. Cell invasion was performed by methylthiazolyldiphenyl-tetrazolium bromide (MTT)-assay.¹³Results were monitored in Thermo Multiscan Ex (ThermoFisher Scientiﬁc Inc, Waltham, MA). Experi- ments were run in triplicate.

Gene expression profiling

To investigate the downstream effect of laeverin-gene silencing in HTR-8/SVneo trophoblast cells, we performed a PCR array to explore 6 biologic pathways that are involved in cell transformation and tumorigenesis (Appendix;Supplemental Table 1).

HTR-8/SVneo trophoblast cells (4-5,710⁵cells) were transfected with Lipofectamine 2000 and 120 pmol siRNA laeverin or siRNA A (control).

Cells were mixed with TRIzol Reagent and RNA isolated by RNeasy Mini kit.

Complementary DNA synthesis and quantitative reverse transcriptionePCR were performed with the use of RT² Proﬁler PCR Array Human Cancer Pathway Finder (PAHS-033A; SABio- sciences Corporation, Frederick, MD).

Actin, beta was used as housekeeping gene. Analysis of fold changes was done by the comparative Ct (

DD

^{Ct) method}

with the integrated web-based software package for the PCR array system.

Statistical analysis

Data were analyzed with IBM SPSS Sta- tistics 21 software (SPSS Inc, Chicago, IL). Continuous variables are presented as mean SE or median (range); cate- goric variables are presented as number (%). Assessment of normality was performed with the Shapiro-Wilk test.

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Differences between groups were tested with the Student t test for parametric variables and the Mann-WhitneyUtest,

c

²test, or F-test for nonparametric variables. A probability value of <.05 was considered signiﬁcant.

R

ESULTS

Phenotype of the study population The baseline demographic and clinical characteristics of the study population that included birth outcomes are shown in theTable. The mean proteinuria level was 5.9 g/L (range, 3.9e9.0 g/L) in women with preeclampsia. None of the women who were included in the study had HELLP (hemolysis, elevated liver enzymes and low-platelets) syndrome.

Two women in the preeclampsia group were delivered by cesarean section because of worsening condition. Two women in the normal group also had cesarean deliveries; one because of breech presentation and another because of placenta previa. None of them were in labor. Four women in each group had vaginal delivery; 3 women in each group had induced labor.

Laeverin ectopically expressed in the trophoblastic cytoplasm in

preeclampsia

Immunoﬂuorescence analysis demonstrated that laeverin is expressed by the villous trophoblasts (Figure 1). In normal placenta, it was membrane-bound

and mainly expressed in plasma membrane (Figure 1, A). Laeverin was expressed more abundantly in the preeclamptic placenta and was localized in the cytoplasm of the villous trophoblast cells (Figure 1, B). In control placentas in which laeverin was replaced by phosphate-buffered saline solution, no speciﬁc labeling was detected in the villous trophoblasts (Figure 1, C).

Molecular mass of laeverin

We estimated the molecular mass of laeverin to be approximately 60 kDa by performing denaturing and reducing SDS-PAGE and Western blot analysis of healthy and preeclamptic placentas (Figure 2). Experiments with our locally designed antibody that was raised against the N- and C-terminal part of laeverin and commercially available antibody that was raised against a peptide mapping within an internal region of laeverin gave the same results.

Laeverin in preeclamptic placentas Immunoelectron microscopy demonstrated that laeverin was expressed in the plasma membrane of trophoblast cells of healthy placentas. It was hardly detectable in the cytosol and was not detectable in the fetal capillaries (Figure 3, B). However, in preeclamptic placentas, laeverin was expressed strongly in the cytoplasm, especially in the microvesicles and in the fetal capillaries (Figure 3, A, C, E, and G). Laeverin was expressed abundantly in microvesicles within the cytoplasm, in the extracellular space, and in areas of focal aggregation of syncytiotrophoblasts (syncytial knots).

Laeverin was not expressed in mito- chondria but was expressed in ER and GA. Experiments with the use of the commercially available laeverin antibody gave same results (Figure 4).

Laeverin silencing affects trophoblast cell migration

Transfection with siRNA against laeverin showed an 11.5% (P ¼.023) reduction in the migration of HTR-8/SVneo trophoblast cells compared with cells that were transfected with scrambled siRNA (control) at the peak of migration, approximately 30 hours after TABLE

Phenotype of the study population

Variable

Preeclampsia (n[6)

Health control

subjects (n[6) Pvalue

Maternal age, y^a 282.35 321.58 .261

Body mass index before delivery, kg/m^2a

28.90.85 29.61.85 .873

Primiparous, n (%) 4 (66.7) 2 (33.3) .567

Mean arterial pressure, mm Hg^a 1313.82 854.15 <.0001 24-hour proteinuria, g/L^a 5.925.10 N/A

Uterine artery pulsatility index (mean of the left and right side)^a

1.230.29 0.690.9 .157

Middle cerebral artery pulsatility index^a

1.200.14 1.380.12 .142 Umbilical artery pulsatility

index^a

1.190.15 0.810.13 .049 Gestational age at delivery, wk^a 341.4 390.48 .005

Cesarean delivery, n (%) 2 (33.3) 2 (33.3) 1

Neonatal birthweight, g^a 2390430 3328207 .055

Placental weight, g^a 43772 62369 .065

5-minute Apgar score^b 8 (6e9) 10 (10e10) .002 Arterial cord blood pH^a 7.270.02 7.250.03 1.0 Arterial cord blood base

excess, mmol/L^a

1.972.06 7.670.58 .069

Venous cord blood pH^a 7.330.02 7.350.02 .343

Venous cord blood base excess, mmol/L^a

2.121.66 4.500.84 .343

Differences between groups were tested with the use of the Studentttest for parametric variables and with the Mann-Whitney Utest orc²test for nonparametric and categorical variables, as appropriate.

N/A, not applicable.

aData are given as meanstandard error;^bData are given as median (range).

Nystad. Laeverin expression is altered in preeclampsia. Am J Obstet Gynecol 2014.

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transfection (Figure 5, A; Supplemental Table 5). Laeverin messenger RNA (mRNA) was 36% reduced in silenced cells (Figure 5, B).

Trophoblast invasion reduced in laeverin-silenced HTR-8/SVneo cells We found that the absorbance (optical density 540) of laeverin-silenced cells

was 0.160.07 and that absorbance at 540 nm of control cells was 0.3 0.11 (P¼.001). Thus, laeverin-silenced cells had a 56.7% reduced ability to invade through Matrigel, compared with control cells (Figure 6). Laeverin mRNA was 80% reduced in silenced cells (data not shown).

Integrin alpha-2, matrix

metalloproteinase 1, and integrin beta-3 genes down-regulated in laeverin-silenced HTR-8/SVneo cells Three genes that are involved in cell transformation and tumorigenesis were shown to be down-regulated by laeverin-silencing at a signiﬁcant level (>4-fold) with the PCR array. Integrin alpha-2, matrix metalloproteinase 1 (MMP1), and integrin beta-3 were down-regulated 39-fold, 36-fold, and 5-fold, respectively (Figure 7). The complete list of genes on the array together with reverse transcriptione PCR results (average threshold cycle [Ct], average difference in cycle number [

D

Ct], and fold-regulation) are given in Supplemental Tables 2-4.

C

OMMENT

Laeverin was ﬁrst reported to be expressed in the cell surface of extravillous trophoblasts obtained from human

third-trimester chorion laeve.²Northern blot analysis showed that laeverin is a placenta-specific protein. It contains a transmembrane domain at the N- terminus and has an amino acid sequence that is homologous with membrane-bound aminopeptidase-N.¹⁴ However, the function of laeverin still is not understood completely. Our immunofluorescence studies on healthy placental tissues demonstrated that laeverin is expressed in the plasma membrane of trophoblast cells, which confirms previousfindings.^4,15However, in preeclamptic placentas, it was localized mainly in the cytoplasm, especially the microvesicles. To our knowledge, this has not been reported previously.

Protein modiﬁcations or cleavage of laeverin could be responsible for its altered placental expression in preeclampsia. However, results of SDS- PAGE and Western blot analysis clearly indicate that laeverin has a molecular mass of 60 kDa (Figure 2) both in preeclamptic and normal placenta. The predicted molecular mass from the amino acid sequence is 113 kDa.¹¹ However, this variance can be explained.

Native laeverin might have cleaved during the puriﬁcation that resulted in 2 identical proteins of 60 kDa, with a total mass of 120 kDa. Furthermore, FIGURE 2

Western blot analysis of laeverin protein in normal and preeclamptic placentas

Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blot analysis withA,locally designed laeverin antibody and B, commercially available laeverin antibody detected a 60-kDa protein. Laeverin protein detected in the placenta of 4 women with preeclampsia (lanes 1-4) and in the placenta of 4 healthy pregnant women (lanes 5-8). Molecular weight marker is shown on the left sideof each figure. Detection of actin protein (43 kDa) was used as loading control (lower part of the figures). Markers used were SeeBlue Plus2 Prestained Standard and Magic Mark XP Western Standard (Invitrogen, Carlsbad, CA).

FIGURE 1

Cross-section of the terminal villi

Trophoblast cells stained with laeverin (green) and counterstained with DAPI II (blue) in A, normal andB,preeclamptic placentas. Laeverin is localized in the plasma membrane of the villous trophoblasts in the normal placenta. In the preeclamptic placenta, laeverin protein is detected in the cytoplasm of the trophoblasts and is more abundant than in normal placenta.

C,In the negative control, no staining of laeverin is detected, but the erythrocytes showed green autofluorescence.²⁷ Original magnifications: A and B,1000; C,600.

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alternate splicing of the laeverin gene may produce 4 protein isoforms with different molecular masses.¹¹ Horie et al⁴detected 3 different bands of 200- 270 kDa, 160 kDa, and 130 kDa in normal placenta instead of 1. This discrepancy could be due to the differences in antibodies and methods that were used for protein puriﬁcation and analysis.

Immunoelectron microscopy demonstrated the expression of laeverin in the fetal capillaries and in the microvesicles within the cytosol of trophoblast cells in preeclamptic placentas. Micro- vesicles in the extracellular space and syncytial knots also expressed laeverin abundantly. Microvesicles and exosomes have been found previously in preeclamptic placentas,¹⁶ and syncytiotrophoblast vesicles have been shown to play a role in the pathophysiologic condition of preeclampsia.¹⁷ Tropho- blastic microvesicles can also be found in the maternal circulation and release cytokines that provoke maternal in- ﬂammatory response.^18,19Microvesicles contain fetal DNA, RNA, and proteins and play an important role in cell communication.²⁰They facilitate intra- cellular transport of proteins and their attachment to the plasma membrane at speciﬁc sites.²¹Colocalization of laeverin together with ER and GA markers indi- cated aberrant processing of laeverin in preeclamptic placentas that may have resulted in massive production of microvesicles. Because the ER and GA in normal placentas did not show accumulation of laeverin, conventional exocytosis might be impaired in preeclamptic placenta.

The laeverin enzyme appears to have a broad spectrum of substrates that can affect cell migration and angiogenesis.²² We found that laeverin silencing reduces migration and invasion of HTR-8/SVneo trophoblast cells. In line with this, Horie et al⁴ have also demonstrated reduced cell invasion in laeverin-silenced human chorionic villous explants cultures.

It has been hypothesized that laeverin plays a role in extravillous trophoblast invasion in cooperation with the chemokine system in the fetomaternal interface.³ Kisspeptin,⁴ angiotensin III, FIGURE 3

Immunoelectron microscopy of ultrathin sections of placentas

Preeclamptic placenta (left column) and healthy placenta (right column). Cross-sections of fetal capillary show red blood cells and protein debris with laeverin (black dots of 5 nm gold particles) inA, preeclamptic placenta andB,unspecific labeling of only red blood cells in normal placenta.C-F, Double labeling with laeverin (5 nm gold) and endoplasmatic reticulum protein disulphide isomerase (PDI) marker (10 nm gold) in preeclamptic and healthy placenta.C,Terminal villi of preeclamptic placenta show a syncytiotrophoblast knot and many microvesicles (arrowheads) andD,no microvesicles in trophoblast cells of healthy placenta.EandF,Magnified sections are depicted as squares.

E,Laeverin and protein disulphide isomerase colocalize in the microvesicles (arrows) in preeclamptic placenta.F,In normal trophoblast cells, no colocalization was detected. Double labeling with laeverin (5 nm gold) and Golgi MG-160 marker (10 nm gold) inG,preeclamptic andH,healthy placenta.

Colocalization was detected in preeclamptic placenta (arrows) but not in cytosol of healthy trophoblast cells. Trophoblast cells of bothC,preeclamptic andD,normal placenta show laeverin localization in the euchromatin of the nucleus.IandJ,Negative control showed no labeling.

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edokinin C, and dynorphin A1-8^9,22,23 are the only known proteins that can be cleaved by laeverin. We looked for other possible interaction partners by performing PCR array on 84 selected genes that are representative of the 6 biologic pathways that are involved in cell transformation and tumorigenesis.

Silencing laeverin had downstream ef- fects on the regulation of the cell adhesion system that is mediated by MMP1 and integrins. MMP1 is expressed by invasive trophoblasts in the ﬁrst-, second-, and third-trimester placentas.²⁴ MMPs are involved in changing cell phenotype from adhesive to a migratory by degrading the extracellular matrix (ECM). They affect cell migration during physiologic processes (such as em- bryonic development, reproduction and tissue remodeling) and in pathologic

conditions (such as cancer metastasis).

Integrins are the main receptors for the ECM²⁵ and are involved in regulating cell adhesion and locomotion. Indeed, trophoblast interaction with the ECM has been shown to be mediated by integrins and MMPs.²⁶ Therefore, it is plausible that laeverin-silenced trophoblasts lose their invasiveness by inter- acting with the cell’s integrin and MMP repertoire.

The molecular link between reduced trophoblast invasion of maternal decidua in the ﬁrst trimester and the development of preeclampsia later in pregnancy is still missing. We have shown previously that laeverin mRNA is increased in preeclamptic placentas.⁶ Our present study shows that laeverin is also increased at protein level and that laeverin silencing reduces trophoblast FIGURE 4

Colocalization immunoelectron microscopy of ultrathin sections of preeclamptic placenta

Commercially available antibody against laeverin (labeled with 5 nm gold) and endoplasmatic reticulum marker protein disulphide isomerase (labeled with 10 nm gold). Laeverin was detectedA,in the endoplasmatic reticulum of trophoblasts (arrows) andB,in microvesicles within the capillaries (arrows). Unspecific labeling of the red blood cells was detected. C, Microvesicles packed with laeverin and protein disulphide isomerase (arrows). Part of section (square inset) inCis magnified andD,shows a close-up of microvesicle with laeverin.

FIGURE 5

Cell migration assays

A,HTR-8/SVneo trophoblast cells in CIM-Plate 16 in the xCelligence system (ACEA Biosciences Inc, San Diego, CA). Graphs show cells that were transfected with small interfering RNA (siRNA) against laeverin. Controls were cells transfected with 2 different types of scrambled nontarget siRNAs (Aand D), untransfected cells, and untransfected cells without serum. Laeverin siRNA silenced cells demonstrated 11.5% reduction of migration. An average of 4 parallels is shown in each graph. Time points (hours) are shown on the x-axis, and cell index is shown on the y-axis.B,Efficacy of laeverin silencing was evaluated with the use of real-time polymerase chain reaction. The siRNA-mediated silencing of laeverin was assessed with the comparative Ct (DDCt) method to determine relative gene expression from quantitative polymerase chain reaction data with actin, beta as an endogenous reference gene. Cells were silenced by laeverin siRNA with messenger RNA reduction of 36%.

Comparison of nontargeting controls (siRNA A and D) to untransfected cells suggests that there is no significant effect of transfection reagent plus siRNA on the cells. Target messenger RNA levels were measured and normalized against actin, beta messenger RNA from samples harvested 55 hours after siRNA transfection of cells. Different experiments are shown on the x-axis, and relative gene expression is shown on the y-axis.

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cell migration and invasion in vitro.

Moreover, it has been shown that endogenous laeverin on the surface of isolated extravillous trophoblastic cells acts to promote their invasion capacity.⁴We found that, in preeclampsia, laeverin is not bound to the trophoblast cell membrane, which indicates a possible deregulation of its physiologic function.

Consequently, one can hypothesize that the production of a deregulated, mal- functioning protein in the preeclamptic placenta might lead to a compensatory increase of laeverin at the mRNA level.

A limitation of our study is that the preeclamptic placentas were delivered earlier compared with the control placentas. However, because term placentas express higher levels of laeverin compared with early (ﬁrst-trimester) placentas,⁴ one would expect to see lower levels of laeverin in preeclamptic placentas that were delivered preterm.

Therefore, the observed differences in laeverin expression are likely to be real

and suggest that the overexpression of laeverin in preeclampsia is associated with the disease process rather than the differences in gestational age. An- other limitation of the study is the small number of placental samples that were used. However, experiments were run in triplicate, and the results were reproducible.

In summary, laeverin, a placenta- speciﬁc protein, appears to be deregulated in preeclampsia that leads to its overexpression and altered subcellular localization in the villous trophoblast.

Whether it could be used potentially as a biomarker of abnormal placentation for prediction and diagnosis of preeclampsia needs further investigation.

We are studying longitudinal changes in laeverin levels in maternal circulation during normal pregnancy and assessing whetherﬁrst- and second-trimester serum laeverin concentration can be used to improve the prediction of preeclampsia in an unselected population. -

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16.Holder BS, Tower CL, Jones CJ, Aplin JD, Abrahams VM. Heightened pro-inﬂammatory effect of preeclamptic placental microvesicles

FIGURE 6

Invasion studies in Boyden chambers

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay of Matrigel invasion of HTR-8/

SVneo trophoblast cells that were transfected with small interfering RNA (siRNA) against laeverin (gray) in Boyden chambers demonstrated 56.7% reduced invasion compared with cells that were transfected with scrambled siRNA A (dark grey). The y-axis represents the absorbance (optical density) at 540 nm (OD 540).

FIGURE 7

Polymerase chain reaction array

Array shows relative expression of 84 genes that were involved in cell transformation and tumorigenesis. Laeverin-silenced HTR-8/SVneo trophoblast cells (x-axis) and controls (y-axis).

The log transformation plot shows relative expression (Log10 [2

ˇ

- DeltaCt]) of each gene (circles) between laeverin-silenced cells and controls.Black linesindicate a 4-fold change in gene expression.

ITGA2, integrin A2 (39-fold); ITGB3, integrin B3 (5-fold); MMP1, matrix metalloprotease 1 (36-fold) are significantly down- regulated.

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24.Huppertz B, Kertschanska S, Demir AY, Frank HG, Kaufmann P. Immunohistochem- istry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta. Cell Tissue Res 1998;291:

133-48.

25.Giancotti FG, Ruoslahti E. Integrin signaling.

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26.Vu TH, Werb Z. Matrix metalloproteinases:

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SUPPLEMENTAL TABLE 1

Polymerase chain reaction array

PCR array catalog #:

Unigene

PAHS-033

Symbol Description Gname RT2 Catalog

Position Refseq

A01 Hs.525622 NM_005163 AKT1 V-akt murine thymoma viral oncogene homolog 1 AKT/PKB PPH00088A

A02 Hs.369675 NM_001146 ANGPT1 Angiopoietin 1 AGP1/AGPT PPH00374A

A03 Hs.583870 NM_001147 ANGPT2 Angiopoietin 2 AGPT2/ANG2 PPH00377E

A04 Hs.552567 NM_001160 APAF1 Apoptotic peptidase activating factor 1 APAF-1/CED4 PPH00752A

A05 Hs.367437 NM_000051 ATM Ataxia telangiectasia mutated AT1/ATA PPH00325B

A06 Hs.370254 NM_004322 BAD BCL2-associated agonist of cell death BBC2/BCL2L8 PPH00075B

A07 Hs.624291 NM_004324 BAX BCL2-associated X protein BCL2L4 PPH00078B

A08 Hs.150749 NM_000633 BCL2 B-cell CLL/lymphoma 2 Bcl-2 PPH00079B

A09 Hs.516966 NM_138578 BCL2L1 BCL2-like 1 BCL-XL/S PPH00082B

A10 Hs.194143 NM_007294 BRCA1 Breast cancer 1, early onset BRCAI/BRCC1 PPH00322E

A11 Hs.599762 NM_001228 CASP8 Caspase 8, apoptosis-related cysteine peptidase ALPS2B/CAP4 PPH00359E

A12 Hs.244723 NM_001238 CCNE1 Cyclin E1 CCNE PPH00131A

B01 Hs.437705 NM_001789 CDC25A Cell division cycle 25 homolog A (S. pombe) CDC25A2 PPH00930A

B02 Hs.19192 NM_001798 CDK2 Cyclin-dependent kinase 2 p33(CDK2) PPH00117E

B03 Hs.95577 NM_000075 CDK4 Cyclin-dependent kinase 4 CMM3/PSK-J3 PPH00118E

B04 Hs.370771 NM_000389 CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) CAP20/CDKN1 PPH00211E

B05 Hs.512599 NM_000077 CDKN2A Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)

ARF/CDK4I PPH00207B

B06 Hs.390736 NM_003879 CFLAR CASP8 and FADD-like apoptosis regulator CASH/CASP8AP1 PPH00333A

B07 Hs.291363 NM_007194 CHEK2 CHK2 checkpoint homolog (S. pombe) CDS1/CHK2 PPH00921B

B08 Hs.517356 NM_030582 COL18A1 Collagen, type XVIII, alpha 1 KNO/KNO1 PPH01141E

B09 Hs.654393 NM_005225 E2F1 E2F transcription factor 1 E2F-1/RBAP1 PPH00136F

B10 Hs.446352 NM_004448 ERBB2 V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)

CD340/HER-2 PPH00209B

Nystad. Laeverin expression is altered in preeclampsia. Am J Obstet Gynecol 2014. (continued)

A

PPENDIX

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Polymerase chain reaction array(continued) PCR array

catalog #:

Unigene

PAHS-033

Position Refseq

B11 Hs.644231 NM_005239 ETS2 V-Ets erythroblastosis virus E26 oncogene homolog 2 (avian)

ETS2IT1 PPH00091B

B12 Hs.244139 NM_000043 FAS Fas (TNF receptor superfamily, member 6) ALPS1A/APO-1 PPH00141B

C01 Hs.533683 NM_000141 FGFR2 Fibroblast growth factor receptor 2 BEK/BFR-1 PPH00391E

C02 Hs.25647 NM_005252 FOS V-fos FBJ murine osteosarcoma viral oncogene

homolog

AP-1/C-FOS PPH00094A

C03 Hs.90708 NM_006144 GZMA Granzyme A (granzyme 1, cytotoxic T-lymphocyte- associated serine esterase 3)

CTLA3/HFSP PPH00314E

C04 Hs.90753 NM_006410 HTATIP2 HIV-1 Tat interactive protein 2, 30kDa CC3/SDR44U1 PPH06957A

C05 Hs.37026 NM_024013 IFNA1 Interferon, alpha 1 IFL/IFN PPH01321A

C06 Hs.93177 NM_002176 IFNB1 Interferon, beta 1, fibroblast IFB/IFF PPH00384E

C07 Hs.160562 NM_000618 IGF1 Insulin-like growth factor 1 (somatomedin C) IGF1A/IGFI PPH00167B

C08 Hs.624 NM_000584 IL8 Interleukin 8 CXCL8/GCP-1 PPH00568A

C09 Hs.644352 NM_181501 ITGA1 Integrin, alpha 1 CD49a/VLA1 PPH00627B

C10 Hs.482077 NM_002203 ITGA2 Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)

BR/CD49B PPH00625E

C11 Hs.265829 NM_002204 ITGA3 Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)

CD49C/GAP-B3 PPH00175A

C12 Hs.694732 NM_000885 ITGA4 Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)

CD49D/IA4 PPH00659E

D01 Hs.436873 NM_002210 ITGAV Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)

CD51/DKFZp686A08142 PPH00628B D02 Hs.643813 NM_002211 ITGB1 Integrin, beta 1 (fibronectin receptor, beta polypeptide,

antigen CD29 includes MDF2, MSK12)

CD29/FNRB PPH00650B

D03 Hs.218040 NM_000212 ITGB3 Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)

CD61/GP3A PPH00178C

D04 Hs.536663 NM_002213 ITGB5 Integrin, beta 5 FLJ26658 PPH00634E

D05 Hs.714791 NM_002228 JUN Jun oncogene AP-1/AP1 PPH00095A

D06 Hs.145442 NM_002755 MAP2K1 Mitogen-activated protein kinase kinase 1 MAPKK1/MEK1 PPH00711B

D07 Hs.599039 NM_006500 MCAM Melanoma cell adhesion molecule CD146/MUC18 PPH00651A

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catalog #:

Unigene

PAHS-033

Position Refseq

D08 Hs.484551 NM_002392 MDM2 Mdm2 p53 binding protein homolog (mouse) HDMX/hdm2 PPH00193E

D09 Hs.132966 NM_000245 MET Met proto-oncogene (hepatocyte growth factor receptor)

AUTS9/HGFR PPH00194A

D10 Hs.83169 NM_002421 MMP1 Matrix metallopeptidase 1 (interstitial collagenase) CLG/CLGN PPH00120B

D11 Hs.513617 NM_004530 MMP2 Matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase)

CLG4/CLG4A PPH00151B

D12 Hs.297413 NM_004994 MMP9 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)

CLG4B/GELB PPH00152E

E01 Hs.525629 NM_004689 MTA1 Metastasis associated 1 Mta-1 PPH01083E

E02 Hs.173043 NM_004739 MTA2 Metastasis associated 1 family, member 2 DKFZp686F2281/MTA1L1 PPH13564A

E03 Hs.700429 NM_014751 MTSS1 Metastasis suppressor 1 DKFZp781P2223/MIM PPH10073A

E04 Hs.202453 NM_002467 MYC V-myc myelocytomatosis viral oncogene homolog (avian)

MRTL/bHLHe39 PPH00100A

E05 Hs.654408 NM_003998 NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1

DKFZp686C01211/EBP-1 PPH00204E E06 Hs.81328 NM_020529 NFKBIA Nuclear factor of kappa light polypeptide gene

enhancer in B-cells inhibitor, alpha

IKBA/MAD-3 PPH00170E

E07 Hs.118638 NM_000269 NME1 Non-metastatic cells 1, protein (NM23A) expressed in AWD/GAAD PPH01314A

E08 Hs.9235 NM_005009 NME4 Non-metastatic cells 4, protein expressed in NDPK-D/NM23H4 PPH01086A

E09 Hs.535898 NM_002607 PDGFA Platelet-derived growth factor alpha polypeptide PDGF-A/PDGF1 PPH00217B

E10 Hs.1976 NM_002608 PDGFB Platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)

PDGF2/SIS PPH00488E

E11 Hs.132225 NM_181504 PIK3R1 Phosphoinositide-3-kinase, regulatory subunit 1 (alpha)

GRB1/p85 PPH00713E

E12 Hs.77274 NM_002658 PLAU Plasminogen activator, urokinase ATF/UPA PPH00796B

F01 Hs.466871 NM_002659 PLAUR Plasminogen activator, urokinase receptor CD87/UPAR PPH00797B

F02 Hs.409965 NM_002687 PNN Pinin, desmosome associated protein DRS/SDK3 PPH19485E

F03 Hs.159130 NM_002880 RAF1 V-raf-1 murine leukemia viral oncogene homolog 1 CRAF/NS5 PPH00227E

F04 Hs.408528 NM_000321 RB1 Retinoblastoma 1 OSRC/RB PPH00228E

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catalog #:

Unigene

PAHS-033

Position Refseq

F05 Hs.654444 NM_002961 S100A4 S100 calcium binding protein A4 18A2/42A PPH01313E

F06 Hs.55279 NM_002639 SERPINB5 Serpin peptidase inhibitor, clade B (ovalbumin), member 5

PI5/maspin PPH00695E

F07 Hs.414795 NM_000602 SERPINE1 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1

PAI/PAI-1 PPH00215E

F08 Hs.349470 NM_003087 SNCG Synuclein, gamma (breast cancer-specific protein 1) BCSG1/SR PPH01051E

F09 Hs.371720 NM_003177 SYK Spleen tyrosine kinase DKFZp313N1010 PPH01639E

F10 Hs.89640 NM_000459 TEK TEK tyrosine kinase, endothelial CD202B/TIE-2 PPH00795B

F11 Hs.492203 NM_198253 TERT Telomerase reverse transcriptase EST2/TCS1 PPH00995E

F12 Hs.645227 NM_000660 TGFB1 Transforming growth factor, beta 1 CED/DPD1 PPH00508A

G01 Hs.494622 NM_004612 TGFBR1 Transforming growth factor, beta receptor 1 AAT5/ACVRLK4 PPH00237B

G02 Hs.164226 NM_003246 THBS1 Thrombospondin 1 THBS/THBS-1 PPH00799E

G03 Hs.522632 NM_003254 TIMP1 TIMP metallopeptidase inhibitor 1 CLGI/EPA PPH00771B

G04 Hs.644633 NM_000362 TIMP3 TIMP metallopeptidase inhibitor 3 HSMRK222/K222 PPH00762A

G05 Hs.241570 NM_000594 TNF Tumor necrosis factor (TNF superfamily, member 2) DIF/TNF-alpha PPH00341E

G06 Hs.521456 NM_003842 TNFRSF10B Tumor necrosis factor receptor superfamily, member 10b

CD262/DR5 PPH00241B

G07 Hs.279594 NM_001065 TNFRSF1A Tumor necrosis factor receptor superfamily, member 1A

CD120a/FPF PPH00346B

G08 Hs.462529 NM_003790 TNFRSF25 Tumor necrosis factor receptor superfamily, member 25

APO-3/DDR3 PPH00349A

G09 Hs.654481 NM_000546 TP53 Tumor protein p53 LFS1/TRP53 PPH00213E

G10 Hs.66744 NM_000474 TWIST1 Twist homolog 1 (Drosophila) ACS3/BPES2 PPH02132A

G11 Hs.563491 NM_017549 EPDR1 Ependymin related protein 1 (zebrafish) EPDR/MERP-1 PPH09305E

G12 Hs.73793 NM_003376 VEGFA Vascular endothelial growth factor A MVCD1/VEGF PPH00251B

H01 Hs.534255 NM_004048 B2M Beta-2-microglobulin B2M PPH01094E

H02 Hs.412707 NM_000194 HPRT1 Hypoxanthine phosphoribosyltransferase 1 HGPRT/HPRT PPH01018B

H03 Hs.523185 NM_012423 RPL13A Ribosomal protein L13a RPL13A PPH01020B

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catalog #:

Unigene

PAHS-033

Position Refseq

H04 Hs.592355 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase G3PD/GAPD PPH00150E

H05 Hs.520640 NM_001101 ACTB Actin, beta PS1TP5BP1 PPH00073E

H06 N/A SA_00105 HGDC Human Genomic DNA Contamination HIGX1A

H07 N/A SA_00104 RTC Reverse Transcription Control RTC

H10 N/A SA_00103 PPC Positive PCR Control PPC

List of genes, fold-changes, and probability values.

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SUPPLEMENTAL TABLE 2 Average Ct

Well Symbol Laeverin knock-out cells Control Group

A01 AKT1 24.53 24.8

A02 ANGPT1 33.86 35

A03 ANGPT2 30.9 30.78

A04 APAF1 25.93 25.84

A05 ATM 27.28 27.22

A06 BAD 24.92 24.5

A07 BAX 22.96 23.25

A08 BCL2 27.14 26.3

A09 BCL2L1 25.08 24.18

A10 BRCA1 25.24 24.77

A11 CASP8 31.14 29.34

A12 CCNE1 27.83 27.19

B01 CDC25A 26.95 26.37

B02 CDK2 23.33 23.12

B03 CDK4 22.3 20.98

B04 CDKN1A 22.79 22.43

B05 CDKN2A 22.82 22.51

B06 CFLAR 26.31 25.42

B07 CHEK2 26.46 25.57

B08 COL18A1 25.83 25.65

B09 E2F1 28.89 29.29

B10 ERBB2 26.51 26.87

B11 ETS2 25.96 25.88

B12 FAS 27.4 27.3

C01 FGFR2 33.2 32.89

C02 FOS 27.57 27.37

C03 GZMA 35 35

C04 HTATIP2 30.45 29.85

C05 IFNA1 32.23 33.53

C06 IFNB1 35 35

C07 IGF1 34.66 34.7

C08 IL8 26.55 26.74

C09 ITGA1 24.87 24.76

C10 ITGA2 28.45 23.11

C11 ITGA3 22.59 23.62

C12 ITGA4 26.88 26.54

D01 ITGAV 24.25 23.26

D02 ITGB1 22.52 21.6

D03 ITGB3 33.25 30.98

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SUPPLEMENTAL TABLE 2 Average Ct(continued)

D04 ITGB5 25.33 24.63

D05 JUN 27.23 27.12

D06 MAP2K1 26.99 25.97

D07 MCAM 24.76 24.73

D08 MDM2 23.36 23.81

D09 MET 24.55 23.69

D10 MMP1 25.75 20.52

D11 MMP2 27.63 27.9

D12 MMP9 28.31 27.64

E01 MTA1 24.19 24.39

E02 MTA2 25.9 26.16

E03 MTSS1 27.33 26.74

E04 MYC 25.6 25.5

E05 NFKB1 25.09 24.52

E06 NFKBIA 24.35 23.75

E07 NME1 21.67 21.52

E08 NME4 23.63 21.87

E09 PDGFA 26.11 26.3

E10 PDGFB 28.1 28.14

E11 PIK3R1 28.48 27.67

E12 PLAU 24.08 22.99

F01 PLAUR 24.78 24.64

F02 PNN 24.07 23.13

F03 RAF1 24.27 23.66

F04 RB1 27.83 26.8

F05 S100A4 27.79 26.94

F06 SERPINB5 35 35

F07 SERPINE1 26.99 27.26

F08 SNCG 26.71 26.72

F09 SYK 35 35

F10 TEK 29.48 27.94

F11 TERT 35 35

F12 TGFB1 23.99 23.41

G01 TGFBR1 29.31 28.76

G02 THBS1 24.89 24.32

G03 TIMP1 21.92 21.42

G04 TIMP3 35 35

G05 TNF 34.96 33.49

G06 TNFRSF10B 24.72 24.95

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SUPPLEMENTAL TABLE 2 Average Ct(continued)

G07 TNFRSF1A 28.29 28.44

G08 TNFRSF25 29.61 28.85

G09 TP53 22.98 23.37

G10 TWIST1 26.6 26.21

G11 EPDR1 27.69 27.27

G12 VEGFA 25 24.61

H01 B2M 20.83 20.77

H02 HPRT1 24.04 23.39

H03 RPL13A 29.54 19.57

H04 GAPDH 18.79 17.31

H05 ACTB 19.88 19.5

H06 HGDC 35 34.71

H07 RTC 21.78 22.99

H08 RTC 21.5 22.86

H09 RTC 21.78 22.87

H10 PPC 18.42 17.82

H11 PPC 18.52 17.73

H12 PPC 18.76 17.62

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SUPPLEMENTAL TABLE 3 2^(-Avg.(Delta(Ct))

Well Symbol Laeverin knock-out Control Group

A01 AKT1 0.076919 0.061462

A02 ANGPT1 0.000119 0.000052

A03 ANGPT2 0.000926 0.000972

A04 APAF1 0.029033 0.029787

A05 ATM 0.011382 0.011486

A06 BAD 0.058744 0.075732

A07 BAX 0.227406 0.18004

A08 BCL2 0.012602 0.021693

A09 BCL2L1 0.052235 0.094312

A10 BRCA1 0.047061 0.062477

A11 CASP8 0.000786 0.00263

A12 CCNE1 0.007796 0.011693

B01 CDC25A 0.01429 0.020707

B02 CDK2 0.175929 0.196621

B03 CDK4 0.359433 0.864344

B04 CDKN1A 0.256466 0.316172

B05 CDKN2A 0.250776 0.30093

B06 CFLAR 0.02228 0.03996

B07 CHEK2 0.02011 0.03586

B08 COL18A1 0.031267 0.033989

B09 E2F1 0.003724 0.002722

B10 ERBB2 0.019492 0.014569

B11 ETS2 0.02843 0.028947

B12 FAS 0.010507 0.010838

C01 FGFR2 0.000188 0.000225

C02 FOS 0.009327 0.010315

C03 GZMA 0.000054 0.000052

C04 HTATIP2 0.001266 0.001847

C05 IFNA1 0.000368 0.000145

C06 IFNB1 0.000054 0.000052

C07 IGF1 0.000068 0.000064

C08 IL8 0.018861 0.015945

C09 ITGA1 0.060808 0.063202

C10 ITGA2 0.005052 0.198592

C11 ITGA3 0.293734 0.139061

C12 ITGA4 0.015075 0.018329

D01 ITGAV 0.093453 0.177765

D02 ITGB1 0.30892 0.564292

D03 ITGB3 0.000183 0.000848

D04 ITGB5 0.044108 0.068928

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SUPPLEMENTAL TABLE 3 2^(-Avg.(Delta(Ct))(continued)

D05 JUN 0.011773 0.01228

D06 MAP2K1 0.013949 0.0272

D07 MCAM 0.065449 0.064392

D08 MDM2 0.172121 0.121974

D09 MET 0.075415 0.132754

D10 MMP1 0.032851 1.191526

D11 MMP2 0.008935 0.007137

D12 MMP9 0.005577 0.008571

E01 MTA1 0.096905 0.081612

E02 MTA2 0.029606 0.023832

E03 MTSS1 0.011011 0.015931

E04 MYC 0.036459 0.037639

E05 NFKB1 0.052116 0.07473

E06 NFKBIA 0.08665 0.126951

E07 NME1 0.555393 0.594031

E08 NME4 0.143637 0.468422

E09 PDGFA 0.025604 0.021651

E10 PDGFB 0.006461 0.006059

E11 PIK3R1 0.004972 0.008382

E12 PLAU 0.10458 0.214459

F01 PLAUR 0.064538 0.06844

F02 PNN 0.105633 0.194843

F03 RAF1 0.091882 0.134911

F04 RB1 0.007782 0.015336

F05 S100A4 0.007992 0.013934

F06 SERPINB5 0.000054 0.000052

F07 SERPINE1 0.013942 0.011173

F08 SNCG 0.016954 0.016164

F09 SYK 0.000054 0.000052

F10 TEK 0.002485 0.006958

F11 TERT 0.000054 0.000052

F12 TGFB1 0.111931 0.160761

G01 TGFBR1 0.002802 0.003939

G02 THBS1 0.059905 0.085591

G03 TIMP1 0.469703 0.636935

G04 TIMP3 0.000054 0.000052

G05 TNF 0.000056 0.000148

G06 TNFRSF10B 0.067447 0.055121

G07 TNFRSF1A 0.005666 0.004922

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SUPPLEMENTAL TABLE 3 2^(-Avg.(Delta(Ct))(continued)

G08 TNFRSF25 0.002268 0.003694

G09 TP53 0.224556 0.1647

G10 TWIST1 0.018237 0.023076

G11 EPDR1 0.008613 0.011097

G12 VEGFA 0.055564 0.069821

H01 B2M 1 1

H02 HPRT1 0.108052 0.163553

H03 RPL13A 0.002381 2.30887

H04 GAPDH 4.096049 11.029787

H05 ACTB 1.932241 2.420406

H06 HGDC 0.000054 0.000064

H07 RTC 0.514533 0.215528

H08 RTC 0.6251 0.235065

H09 RTC 0.514609 0.234047

H10 PPC 5.314673 7.769474

H11 PPC 4.95624 8.213203

H12 PPC 4.183476 8.865592

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SUPPLEMENTAL TABLE 4 Fold reg

Well Symbol Laeverin knock-out cells

A01 AKT1 1.2515

A02 ANGPT1 2.2862

A03 ANGPT2 -1.049

A04 APAF1 -1.026

A05 ATM -1.0091

A06 BAD -1.2892

A07 BAX 1.2631

A08 BCL2 -1.7215

A09 BCL2L1 -1.8055

A10 BRCA1 -1.3276

A11 CASP8 -3.3446

A12 CCNE1 -1.4999

B01 CDC25A -1.449

B02 CDK2 -1.1176

B03 CDK4 -2.4047

B04 CDKN1A -1.2328

B05 CDKN2A -1.2

B06 CFLAR -1.7935

B07 CHEK2 -1.7832

B08 COL18A1 -1.087

B09 E2F1 1.3682

B10 ERBB2 1.3379

B11 ETS2 -1.0182

B12 FAS -1.0315

C01 FGFR2 -1.1974

C02 FOS -1.1059

C03 GZMA 1.0375

C04 HTATIP2 -1.4591

C05 IFNA1 2.5468

C06 IFNB1 1.0375

C07 IGF1 1.0659

C08 IL8 1.1829

C09 ITGA1 -1.0394

C10 ITGA2 -39.309

C11 ITGA3 2.1123

C12 ITGA4 -1.2159

D01 ITGAV -1.9022

D02 ITGB1 -1.8267

D03 ITGB3 -4.6435

D04 ITGB5 -1.5627

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SUPPLEMENTAL TABLE 4 Fold reg(continued)

D05 JUN -1.0431

D06 MAP2K1 -1.95

D07 MCAM 1.0164

D08 MDM2 1.4111

D09 MET -1.7603

D10 MMP1 -36.2703

D11 MMP2 1.252

D12 MMP9 -1.5369

E01 MTA1 1.1874

E02 MTA2 1.2423

E03 MTSS1 -1.4469

E04 MYC -1.0324

E05 NFKB1 -1.4339

E06 NFKBIA -1.4651

E07 NME1 -1.0696

E08 NME4 -3.2611

E09 PDGFA 1.1826

E10 PDGFB 1.0664

E11 PIK3R1 -1.6861

E12 PLAU -2.0507

F01 PLAUR -1.0605

F02 PNN -1.8445

F03 RAF1 -1.4683

F04 RB1 -1.9707

F05 S100A4 -1.7435

F06 SERPINB5 1.0375

F07 SERPINE1 1.2478

F08 SNCG 1.0488

F09 SYK 1.0375

F10 TEK -2.7997

F11 TERT 1.0375

F12 TGFB1 -1.4362

G01 TGFBR1 -1.4057

G02 THBS1 -1.4288

G03 TIMP1 -1.356

G04 TIMP3 1.0375

G05 TNF -2.6713

G06 TNFRSF10B 1.2236

G07 TNFRSF1A 1.1513

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SUPPLEMENTAL TABLE 4 Fold reg(continued)

G08 TNFRSF25 -1.6286

G09 TP53 1.3634

G10 TWIST1 -1.2653

G11 EPDR1 -1.2884

G12 VEGFA -1.2566

H01 B2M 1

H02 HPRT1 -1.5136

H03 RPL13A -969.6478

H04 GAPDH -2.6928

H05 ACTB -1.2526

H06 HGDC -1.1759

H07 RTC 2.3873

H08 RTC 2.6593

H09 RTC 2.1987

H10 PPC -1.4619

H11 PPC -1.6571

H12 PPC -2.1192

P Placentalexpressionofaminopeptidase-Q(laeverin)anditsroleinthepathophysiologyofpreeclampsia Research

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Placental expression of aminopeptidase-Q (laeverin) and its role in the pathophysiology of preeclampsia

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